This invention generally relates to imaging objects or particles for purposes of detection and analysis, and more specifically, to a system and method for analyzing the spectral composition, spatial characteristics, and temporal behavior of objects, such as cells, which may be in motion.
Development of new drugs to treat diseases and other medical problems is an expensive and time-consuming process. The efficiency of the drug discovery process is hindered by the limitations of current cell and particle analysis technology. These limitations affect drug discovery at every stage, including: target discovery, target validation, screening, lead optimization, and clinical development. Cell and particle analysis technology is an important aspect of this problem, because one of the goals of the drug discovery process is to understand the biological effect of potential drug compounds on targeted cell types and the collateral effects on other cell types.
In many cases, fluorescent tags are used to label both potential drug compounds and various cellular components in order to detect and analyze binding interactions in both in vitro and in vivo assays. In order to distinguish different compounds and biological targets, each can be labeled with a different fluorescent tag. Therefore, the number of compounds and targets that can be simultaneously studied is limited by the number of colors that can be discriminated. Binding interactions in biological systems are dynamic processes that require evaluation at different points in time. Such interactions can occur over intervals of only a few microseconds. Hence, the ability to discriminate the time sequence of events in an assay is a function of the speed with which repeated fluorescent measurements can be made.
The interactions between compounds and biological targets are preferably studied in an intact cell, in order to detect both beneficial and adverse effects. These effects are often evidenced by the presence or absence of fluorescence in different locations within or around the cell or by changes in cell morphology. Accordingly, the ability to detect the biological activity of a drug candidate is also a function of the spatial resolution of the detection system. Therefore, an ideal system for drug discovery should possess high spectral, temporal, and spatial resolution. An ideal system should further possess high sensitivity to detect low concentrations of biological targets and faint fluorescent signals. Finally, an ideal system would have high throughput to allow the rapid analysis of large compound libraries and numerous biological targets within different cell types.
Rudimentary time-series images of stationary cells can be acquired with a limited set of three or four colors using existing frame-based imaging technology. The measurement frequency of most video imaging systems is approximately 30 Hz, which limits their ability to measure transients that occur in less than about 100 ms. In some cases, the cells under study may be moving, as in microfluidic xe2x80x9clab on chipxe2x80x9d systems. In order to prevent image blurring when the cell or objects under study are in motion, the exposure time must be kept very short, which reduces sensitivity.
Accordingly, it will be apparent that an improved technique is desired that resolves the limitations in analyzing the spectra, images, and kinetics of both stationary and moving cells imposed by conventional imaging systems. In addition, a new approach developed to address these problems in the prior art should also have application to the analysis of other types of objects besides cells and should be capable of implementation in different configurations to meet the specific requirements of disparate applications of this technology.
The present invention is directed to an imaging system that is adapted to determine one or more characteristics of an object from an image of the object. There can be relative movement between the object and the imaging system, and although it is contemplated that either (or both) may be in motion, the object will preferably move while the imaging system is fixed in position. In addition, it should also be understood that while much of the discussion and the claims that follow recite xe2x80x9can object,xe2x80x9d it is likely that the present invention will preferably be used with a plurality of objects and is particularly useful in connection with a stream of objects or objects moving within a substrate; e.g., in narrow capillaries. Also, it should be understood that as used herein and in the following claims, the terms xe2x80x9cimagexe2x80x9d and xe2x80x9cimagingxe2x80x9d are broadly applied and are intended to generally refer to the light from an object or objects that is directed onto a surface of a detector; thus, these terms are intended to encompass light from an object or objects that is diffused, dispersed, or blurred on the surface of a detector, as well as light from an object or objects that is focussed onto the surface of the detector, and light from an object or objects that is divided into one or more spectral components incident on the surface of the detector.
The present invention is directed to a method and apparatus for the spectral, spatial, and temporal analysis of cells for purposes of drug discovery and other applications. To achieve such functionality, the present invention rapidly collects image data from moving cells over time. These data can include simultaneous spatial and spectral images covering a wide bandwidth at high resolution. Further, the present invention preserves the spatial origin of the spectral information gathered from the object(s).
In addition, the present invention offers considerable advantages over prior art systems employed for cell and particle analysis. Some of these advantages arise from the novel application of a time delay integration (TDI) detector that produces an output signal in response to the images of cells and other objects that are directed on the TDI detector. The TDI detector that is used in the various embodiments of the present invention preferably comprises a rectangular charge-coupled device (CCD) that employs various specialized pixel read out algorithms, as explained below.
Standard, non-TDI CCD arrays are commonly used for imaging in cameras. In a standard CCD array, photons that are incident on a pixel produce charges that are trapped in the pixel. After image acquisition, the photon charges from each pixel are read out of the detector array by shifting the charges into an output capacitor, producing a voltage proportional to the charge. Between pixel readings, the capacitor is discharged and the process is repeated for every pixel on the chip. During the readout, the array must be shielded from any light exposure to prevent charge generation in the pixels that have not yet been read.
In a TDI detector comprising a CCD array of physical pixels, the CCD array remains exposed to the light as the pixels are read out. The projection of an image on the array of physical pixels generates a pixelated signal. Readout of this signal occurs one row at a time, e.g., from the top to the bottom of the array. Once a first row is read out, the signal pixels in the remaining rows are shifted by one physical pixel in the direction of the row that has just been read. If the object being imaged onto the array moves in synchrony with the motion of the signal pixels, light from the object is integrated without image blurring for the duration of the TDI detector""s total readout period. The signal strength produced by a TDI detector increases linearly with the integration period, which is proportional to the number of physical TDI pixel rows, but the noise increases only as the square root of the integration period, resulting in an overall increase in the signal-to-noise ratio by a factor equal to the square root of the number of rows.
If the image of the object moves synchronously with the pixelated signal (in the same direction and with the same speed), light forming each portion of the image is detected in the same portion of the pixelated signal over time, regardless of the motion. Conversely, if the image of the object moves asynchronously relative to the pixelated signal, (at a different speed and/or in a different direction), light forming each portion of the image at later times will not be detected in the same portion of the pixelated signal that corresponded to the image portion at an earlier time. By intentionally desynchronizing the motion of the pixelated signal on the TDI detector from the motion of the image, temporally distinct pixelated signals are produced. The desynchronization can result from a difference in the speed of the image relative to the signal and/or a difference in the direction of motion between the two. In this manner, time-resolved measurements of morphology and spectral emission characteristics are performed.
In the present invention, there are four entities that may be in motion. These include the object being imaged, the image of the object projected on the detector, the detector itself, and the signal generated by the image on the detector. Any movement of the object relative to the detector results in movement of the image across the detector. However, movement of the object is not required in the present invention. Depending on the embodiment of the invention, there may or may not be relative motion between the image and the detector. TDI imaging, unlike other imaging methods, involves the movement of the signal across the detector while the measurement is being performed. However, the signal need not move in synchrony with the image of the object. In the present invention, the velocity of signal motion is a controllable parameter that can be adjusted in order to measure various features of the object being imaged. The signal can be made to move faster, slower, or in a different direction than the image, which may or may not itself be moving. Further, the movement of the signal can be changed dynamically during the measurement. The nature of the asynchrony in part determines the features of an object or objects that can be measured.
In several embodiments of the present invention, relative movement will exist between the object being imaged and the imaging system, and in most cases, it will be more convenient to move the object than to move the imaging system. However, it is also contemplated that in some cases, the object may remain stationary, and the imaging system move relative to it. As a further alternative, both the imaging system and the object may be in motion, but in different directions or at different rates. Regardless of whether there is relative movement between the object and the imaging system, there will be a movement of the signal across the detector. The synchrony of signal movement is preferably adjusted by changing either the speed of the object, the speed of the signal, or the direction of the signal.
Another adjustable parameter in the present invention is the continuity of signal generation. In some embodiments of the invention, the signal from the object is detected continuously. An exemplary application of continuous detection would be the imaging of a cell containing a chemiluminescent substrate that constantly emits light. Another example is a cell illuminated by a continuous-wave laser or arc lamp, forming either a scatter, absorption, or fluorescence image on the detector. In other embodiments of the present invention, the signal from the object is detected in a discontinuous fashion. For example, a discontinuous detection occurs if a cell is illuminated by a pulsed or modulated laser, forming either transient scatter, absorption, or fluorescence images on the detector. Another example of discontinuous detection occurs if a chemiluminescent cell is imaged via a shuttered or gated TDI detector. Signal continuity, when controlled in combination with the synchrony of signal readout, gives rise to various modes of operation of the present invention.